Thermophilic endo-glucanase and uses thereof

ABSTRACT

A novel thermophilic endo-glucanase, nucleic acid encoding the endo-glucase, and uses thereof in converting ligocellulosic material to fermentable sugars.

RELATED APPLICATIONS

This application is a divisional application of and claims priority toU.S. Utility application Ser. No. 12/541,322, filed on Aug. 14, 2009(now U.S. Pat. No. 7,927,856), which in turn claims priority to U.S.Provisional Application No. 61/089,100, filed on Aug. 15, 2008, thecontents of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

Given the shortage of conventional energy sources (e.g., oil), manyefforts have been spent on exploring alternative energy sources. Amongthem, plant biomass is of particular interest as it is renewable.

Plant mass contains a high amount of cellulose, a starting material formaking biofuel. To convert cellulose to biofuel, it is first degraded tofermentable sugars, such as cellobiose and glucose, by the cellulolyticsystem of microorganisms. This system includes three major types ofhydrolases, i.e., endoglucanases (EC 3.2.1.4), exoglucanases (EC3.2.1.91), and β-glucosidases (EC 3.2.1.21). Many cellulolytic enzymeshave been isolated from various microorganisms, most of which exhibitoptimal enzymatic activities at temperature below 50° C. As such, theseenzymes do not efficiently degrade cellulose, the crystalline structureof which is typically destroyed above 50° C. Thus, there is a need foran efficient and thermophilic cellulolytic enzyme.

SUMMARY OF THE INVENTION

This invention is based on, at least in part, the discovery of a novelthermophilic endoglucanase isolated from Geobacillus sp. 70PC53, i.e.,CelA. Shown in FIG. 1 are the polypeptide sequence of CelA and its cDNAsequence (SEQ ID NOs: 1 and 2, respectively)

Accordingly, one aspect of this invention features an isolatedpolypeptide containing a sequence exhibiting at least 70% (e.g., 80%,90%, 95%, or 99%) amino acid identity to SEQ ID NO:1 as determined bythe BLAST algorithm.

This invention also encompasses (i) an isolated nucleic acid including anucleotide sequence that encodes the polypeptide described above, and(ii) a host cell containing such an isolated nucleic acid. In oneexample, the nucleotide sequence is SEQ ID NO: 2. The isolated nucleicacid of this invention can be an expression vector, in which thenucleotide sequence is operably linked to a suitable promoter sequence(i.e., a sequence capable of initiating transcription in a host cell).

The term “isolated polypeptide” or “isolated nucleic acid” used hereinrefers to a polypeptide or nucleic acid substantially free fromnaturally associated molecules, i.e., the naturally associated moleculesconstituting at most 20% by dry weight of a preparation containing thepolypeptide or nucleic acid. Purity can be measured by any appropriatemethod, e.g., column chromatography, polyacrylamide gel electrophoresis,and HPLC.

Also within the scope of this invention is a method of producing afermentable sugar from a ligocellulosic material. This method includes(i) providing a multi-enzyme composition containing the polypeptidedescribed above, an exo-glucanase, and a β-glucosidase, and (ii)contacting the multi-enzyme composition with a lignocellulosic materialto produce a fermentable sugar, e.g., glucose, xylose, arabinose,galactose, mannose, rhamnose, sucrose, or fructose. The fermentablesugar can be converted to a fermentation product, such as alcohol, bymicroorganism fermentation or enzyme treatment. Examples of thelignocellulosic material used in this method include, but are notlimited to, orchard prunings, chaparral, mill waste, urban wood waste,municipal waste, logging waste, forest thinnings, short-rotation woodycrops, industrial waste, wheat, wheat straw, oat straw, rice straw,barley straw, rye straw, flax straw, soy hulls, rice hulls, oat hulls,sugar cane, corn, corn stover, corn stalks, corn gluten feed, corn cobs,corn husks, corn kernel, fiber from kernels, prairie grass, gamagrass,foxtail, sugar beet pulp, citrus fruit pulp, seed hulls, cellulosicanimal wastes, lawn clippings, cotton, seaweed, trees, shrubs, grasses,sugar cane bagasse, products and by-products from wet or dry milling ofgrains, municipal solid waste, waste paper, yard waste, herbaceousmaterial, agricultural residues, forestry residues, municipal solidwastes, waste paper, pulp, paper mill residues, branches, bushes, canes,corn, corn husks, energy crops, forests, fruits, flowers, grains,grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings,shrubs, switch grasses, trees, vegetables, fruit peels, vines, sugarbeet pulp, wheat midlings, oat hulls, hard and soft woods, organic wastematerials generated from agricultural processes, forestry wood waste, orcombinations thereof.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of an example, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are first described.

FIG. 1 is a diagram showing the polypeptide and coding sequences of CelA(SEQ ID NO:1 and SEQ ID NO:2, respectively).

FIG. 2 is a diagram showing the optimal reaction temperature (panel A)and thermostability (panel B) of CelA.

FIG. 3 is a diagram showing the optimal pH (panel A) and pH stability(panel B) of CelA.

FIG. 4 is a chart showing effects of various chemicals on theendo-glucanase activity of CelA.

DETAILED DESCRIPTION OF THE INVENTION

Described herein is an isolated polypeptide including an amino acidsequence at least 70% identical to that of CelA (SEQ ID NO:1) or a partthereof having at least 20 (e.g., 30, 50, 80, 100, 150, 200, 250, 300,and 350) contiguous amino acids. See Ng et al., Extremophiles 13:425-435(2009). This polypeptide is an thermophilic endo-glucanase thathydrolyzes the 1,4-beta-D-glycosidic linkages in cellulose, lichenin,and cereal beta-D-glucans.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of the invention. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

The isolated polypeptide can be prepared by purification from a suitablemicroorganism, e.g., Geobacillus. It also can be prepared viaconventional recombinant technology. An example follows. A DNA fragmentencoding CelA can be prepared by polymerase chain reaction fromGeobacillus cells and cloned into an expression vector. Upon insertion,the CelA-encoding fragment is operably linked to a suitable promotercontained in the expression vector. The resultant DNA construct is thenintroduced into suitable host cells (e.g., E. coli cells, yeast cells,insect cells, and mammalian cells) for expression of CelA, which can bepurified from the cells by conventional methods.

To make a functional equivalent of CelA, which is also within the scopeof this invention, one or more conservative amino acid substitutions canbe introduced into SEQ ID NO:1 without disrupting its endo-glucanaseactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in SEQ IDNO: 1 is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, mutations can be introducedrandomly along all or part of SEQ ID NO: 1, such as by saturationmutagenesis, and the resultant mutants can be screened for theendoglucanase activity to identify mutants that retain the activity asdescried below in the Example section below.

Fusion protein technology can be applied to improve expressionefficiency and facilitate purification of the polypeptide of thisinvention. To prepare a fusion protein containing CelA, a DNA fragmentencoding this endo-glucanase can be linked to another DNA fragmentencoding a fusion partner, e.g., glutathione-s-transferase (GST), 6×-Hisepitope tag, or M13 Gene 3 protein. The resultant fusion nucleic acidexpresses in suitable host cells a fusion protein that can be isolatedby methods known in the art. The isolated fusion protein can be furthertreated, e.g., by enzymatic digestion, to remove the fusion partner andobtain the recombinant polypeptide of this invention.

Also described herein is an isolated nucleic acid encoding thepolypeptide of this invention. A nucleic acid refers to a DNA molecule(e.g., a cDNA or genomic DNA), an RNA molecule, or a DNA/RNA analog,which can be synthesized from nucleotide analogs. In one example, thenucleic acid of this invention is an expression vector in which a DNAfragment encoding the polypeptide is operably linked to a suitablepromoter.

As used herein, the term “promoter” refers to a nucleotide sequencecontaining elements that initiate the transcription of an operablylinked nucleic acid sequence in a desired host microorganism. At aminimum, a promoter contains an RNA polymerase binding site. It canfurther contain one or more enhancer elements which, by definition,enhance transcription, or one or more regulatory elements that controlthe on/off status of the promoter. When E. coli is used as the hostmicroorganism, representative E. coli promoters include, but are notlimited to, the β-lactamase and lactose promoter systems (see Chang etal., Nature 275:615-624, 1978), the SP6, T3, T5, and T7 RNA polymerasepromoters (Studier et al., Meth. Enzymol. 185:60-89, 1990), the lambdapromoter (Elvin et al., Gene 87:123-126, 1990), the trp promoter(Nichols and Yanofsky, Meth. in Enzymology 101:155-164, 1983), and theTac and Trc promoters (Russell et al., Gene 20:231-243, 1982). Whenyeast is used as the host microorganism, exemplary yeast promotersinclude 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphatedehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter,galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter.Promoters suitable for driving gene expression in other types of cellsare also well known in the art.

A vector refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. The vector can becapable of autonomous replication or integrate into a host DNA. Examplesof the vector include a plasmid, cosmid, or viral vector. The vector ofthis invention includes a nucleotide sequence encoding CelA in a formsuitable for expression of the nucleic acid in a host cell. Preferablythe vector includes one or more regulatory sequences operatively linkedto the encoding sequence. A “regulatory sequence” includes promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those that direct constitutiveexpression of a nucleotide sequence, as well as tissue-specificregulatory and/or inducible sequences. The design of the expressionvector can depend on such factors as the choice of the host cell to betransformed, the level of expression of protein desired, and the like.The expression vector can be introduced into host cells to produce thepolypeptide of this invention.

Also within the scope of this invention is a host cell that contains theabove-described nucleic acid. Examples include E. coli cells, insectcells (e.g., using baculovirus expression vectors), yeast cells, plantcells, or mammalian cells. See e.g., Goeddel, (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.To produce a polypeptide of this invention, one can culture a host cellin a medium under conditions permitting expression of the polypeptideencoded by a nucleic acid of this invention, and purify the polypeptidefrom the cultured cell or the medium of the cell. Alternatively, thenucleic acid of this invention can be transcribed and translated invitro, for example, using T7 promoter regulatory sequences and T7polymerase.

Further described herein is a method of converting lignocellulosicmaterial to fermentable products (e.g., fermentable sugars) using amulti-enzyme composition containing the endo-glycanase described hereinand other cellulolytic enzymes, such as exo-glucanase and β-glucosidase.See, e.g., US Application Nos. 20070238155 and 20070250961. The term“cellulolytic enzyme” refers to an enzyme that hydrolyzes cellulose (apolysaccharide consisting of glucose units) into smaller sugar units.See Gilbert H J, Hazlewood G P, 1993 J Gen Microbiol 139:187-194;Olimiya K et al. 1997 Biotechnol Genet Eng Rev. 14:365-414. See also,e.g., US Application 2007016805. This multi-enzyme composition can beobtained from, e.g., a microbial, a plant, or a combination thereof, andwill contain enzymes capable of degrading lignocellulosic material. Inaddition to the cellulolytic enzymes mentioned above, it can furtherinclude cellobiohydrolases, endoglucanase, beta.-glucosidases),hemicellulases (such as xylanases, including endoxylanases, exoxylanase,and beta-xylosidase), ligninases, amylases, alpha-arabinofuranosidases,alpha-glucuronidases, alpha-glucuronidases, arabinases, glucuronidases,proteases, esterases (including ferulic acid esterase and acetylxylanesterase), lipases, glucomannanases, or xylogluconases.

As used herein the term “lignocellulosic material” refers to materialscontaining cellulose and/or hemicellulose. Generally, these materialsalso contain xylan, lignin, protein, and carbohydrates, such as starchand sugar. Lignocellulose is found, for example, in the stems, leaves,hulls, husks, and cobs of plants or leaves, branches, and wood of trees.The process of converting a complex carbohydrate (such as starch,cellulose, or hemicellulose) into fermentable sugars is also referred toherein as “saccharification.” Fermentable sugars, as used herein, referto simple sugars, such as glucose, xylose, arabinose, galactose,mannose, rhamnose, sucrose and fructose. Lignocellulosic material caninclude virgin plant biomass and/or non-virgin plant biomass such asagricultural biomass, commercial organics, construction and demolitiondebris, municipal solid waste, waste paper, and yard waste. Common formsof lignocellulosic material include trees, shrubs and grasses, wheat,wheat straw, sugar cane bagasse, corn, corn husks, corn kernel includingfiber from kernels, products and by-products from milling of grains suchas corn, rice, wheat, and barley (including wet milling and drymilling), as well as municipal solid waste, waste paper, and yard waste.The lignocellulosic material can also be, but is not limited to,herbaceous material, agricultural residues, forestry residues, and papermill residues. “Agricultural biomass” includes branches, bushes, canes,corn and corn husks, energy crops, forests, fruits, flowers, grains,grasses, herbaceous crops, leaves, bark, needles, logs, roots, saplings,short rotation woody crops, shrubs, switch grasses, trees, vegetables,fruit peels, vines, sugar beet pulp, wheat midlings, oat hulls, hard andsoft woods (not including woods with deleterious materials), organicwaste materials generated from agricultural processes including farmingand forestry activities, specifically including forestry wood waste, ora mixture thereof.

The fermentable sugar produced in the method described above can beconverted to useful value-added fermentation products via enzymetreatment or chemical reaction. Examples of the fermentation productinclude, but are not limited to amino acids, vitamins, pharmaceuticals,animal feed supplements, specialty chemicals, chemical feedstocks,plastics, solvents, fuels, or other organic polymers, lactic acid, andethanol, including fuel ethanol. Specific value-added fermentationproducts that may be produced by the methods of the invention include,but not limited to, biofuels (including ethanol and butanol); lacticacid; plastics; specialty chemicals; organic acids, including citricacid, succinic acid and maleic acid; solvents; animal feed supplements;pharmaceuticals; vitamins; amino acids, such as lysine, methionine,tryptophan, threonine, and aspartic acid; chemical feedstocks. Thefermentable sugar can also be used for culturing microbes that producefermentation products, e.g., industrial enzymes, such as proteases,cellulases, amylases, glucanases, lactases, lipases, lyases,oxidoreductases, transferases and xylanases.

The invention also provides a method of producing energy fromlignocellulosic material. This method include providing the multi-enzymecomposition described above; contacting the composition with thelignocellulosic material to produce a fermentable product; fermentingthe fermentable product to produce a combustible fermentation product,and combusting the combustible fermentation product to produce energy.This method can be performed in a bioreactor that contains all necessarycomponents and may preferably be configured for anaerobic growth ofmicroorganisms. Methods for making and using bioreactors are known inthe art. See, e.g., US Application 20080131958.

The polypeptide and composition described above can also be used in thepaper and pulp industry. For example, they can be used in the deinkingand refining of recycled paper. In this application, utilizing athermostable cellulase, i.e. having optimal activity at temperatures of65° C. or higher versus having optimal activity at room temperature,could reduce the amount of enzyme used per ton of paper substantially,and reduce the time of exposure to the enzyme needed to increase thebrightness of the paper. Reducing the concentration of enzyme and thetime of exposure to the enzyme in the refining process, correspondinglyand desirably reduces the reaction of the cellulase on the fibrilsthemselves and processing costs.

The polypeptide of this invention has additional industrial applicationswhere high temperatures are needed (e.g., clarification of fruitjuices). Given its high thermostability (see the Example below), thispolypeptide can function under high temperatures with no need toincrease its amount. The polypeptide, in combination with other enzymes,can be used, with enhanced yields, in extracting juice from fruits, orextracting juice or soup flavorings from vegetables. In combination withprotease, it can be used to dissociate dried seaweed, which is thenfermented with alcohol to produce vinegar. The polypeptide, mixed withother enzymes, can also serve as a dough conditioner in the bakingindustry. See, e.g., U.S. Pat. No. 6,602,700

Moreover, the polypeptide of this invention can also be used in thetextile industry. It can be used to brighten and soften cotton fabricsby removing microfibers on the surface, which causes a dull look ofclothes. More specifically, it can be included as an additive informulating enzyme-containing detergents for soil removal, fabricsoftening, and color brightening. For example, it can be used as areplacement to pumice in producing blue jeans having a “stone-washed”effect. Enzyme treatment causes less damage to the jean fabric thanlengthy exposure to pumice. See U.S. Pat. Nos. 5,232,851, 5,677,151,6,451,063, and 7226773.

In another aspect, the present invention provides a transgenic plant,the genome of which is augmented with a recombinant polynucleotideencoding a polypeptide of this invention operably linked to a promotersequence. The polynucleotide is optimized for expression in the plantand the polypeptide is produced at a level greater than 5% total solubleprotein, greater than 10% total soluble protein or greater than 20%total soluble protein. The polypeptide may be expressed constitutivelyor tissue-specifically. For example, it may be expressed in a planttissue selected from the group consisting of stems and leaves. It mayalso be expressed in a targeted sub-cellular compartment or organelle,such as apoplast, chloroplast, cell wall, or vacuole. The plant may be amonocotyledonous plant or a dicotyledonous plant. In certainembodiments, the plant is a crop plant. The plant may be selected fromthe group consisting of corn, switchgrass, sorghum, miscanthus,sugarcane, poplar, pine, wheat, rice, soy, cotton, barley, turf grass,tobacco, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.Methods for making transgenic plants are well known in the art.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific example is, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever. All publications cited herein areincorporated by reference.

Cloning, Preparation, and Characterization of Geobacillus sp. 70PC53Endo-Glucanase

Isolation of Geobacillus sp. 70PC53 from Rice Straw Compost

A mixture of pig manure, pigbone powder, rice hull, and rice straw at aweight ratio of 3:1:12:6, was incubated at 55-70° C. to produce a ricestraw compost. The rice straw compost (2 g) was mixed with rice hull (1g) in 100 ml of minimal requirement (MR) medium (containing in one literwater, 1.4 g (NH₄)₂SO4, 2.0 g KH₂PO4, 0.34 g CaCl2.2H₂O, 0.30 gMgSO4.7H₂O, 5 mg FeSO4.7H₂O, 1.6 mg MnSO4.H₂O, 1.4 mg ZnSO₄.7H₂O and 2.0mg CoCl₂.6H₂; see Mandels et al., J. Bacteriol 73:269-278, 1957)supplemented with 1.0 g carboxylmethyl cellulose (CMC). The mixture wasincubated at 65° C. overnight (16 h) and its supernatant (100 ll) wasplaced on an MR-CMC plate (containing the MR medium supplemented with 1%CMC) and incubated at the same temperature for 24 h. Bacterial coloniesgrown on the MR-CMC plate were isolated, re-inoculated on a fresh MR-CMCplate, and incubated for a suitable period. The MR-CMC plate was thenstained with a solution containing 0.2% Congo red solution and destainedwith a NaCl solution (1N). Bacterial colonies around which clear zonesdisplayed, were chosen for further characterization. Identity of eachisolated bacterial strain was analyzed by the 16S rDNA sequencing methoddescribed in Kim et al., Int. J. Syst. Evol. Microbiol. 50:1641-1647,2000, using universal primers 27f: 5′-AGAGTTTGATCCTGGCT CAG-3′ and1497r: 5′-AAGTCGTAACAAGGTAACC-3′.

The 16S rDNA analysis indicates that among all selected bacterialstrains, over 100 are Geobacillus sp. strains. Strain 70PC53 wasidentified for exhibited high cellulolytic activity as evidenced bygeneration of a large-sized clear zone on the MR-CMC plate. Otherfeatures of this strain are described in Ng et al., Extremophiles13:425-435, 2009.

Cloning of a Novel Endo-Glucanase CelA from Geobacillus sp. 70PC53

Genomic DNA was purified from Geobacillus sp. 70PC53, partially digestedwith EcoRI, and resolved on a 0.8% agarose gel. DNA fragments rangingfrom 3 to 10 kb were recovered from the gel, cloned into pBluescriptIISK(+) (Stratagene, USA), and introduced into E. coli DH5a cells togenerate a genomic library. Individual transformants were grown onLuria-Bertani (LB) agar plates containing 1.0% (w/v) CMC and 100 lg/mlampicillin. Colonies that exhibited cellulolytic enzyme activity wereidentified following the method described above. Plasmid DNA of eachpositive clone was isolated and digested with EcoRI and HindIII torelease the DNA fragment inserted therein. The DNA fragment was theninserted into the pBluescript vector, and subjected to DNA sequencing.One DNA fragment (1.104 bp) was found to encode a endo-glucanase(designated CelA endo-glucanase) having the amino acid sequence of SEQID NO:1.

Preparation of Recombinant CelA

The DNA fragment encoding CelA endo-glucanase was amplified by PCR,using forward primer, 5′-GGGAACATATGGTGA AAAAAGCT TTTCTGCCCGTG-3′ (NdeIsite underlined) and reverse primer, 5′-CGCCCCTCGAGCTCTTTGAACAAACGTTTCCCT-3′(XhoI site underlined). The PCR product was inserted into thepET-20b(+) vector (encoding a His-tag) and introduced into E. colistrain Rosetta C41. A positive transformant was inoculated into 100 mlLB medium supplemented with 100 mg/ml ampicillin and cultured at 37° C.in a rotary shaker (150 rpm). When the OD₆₀₀ value of the cell culturereached 0.4 to 0.6, 1 mM of iso-propyl-b-thiogalactopyranoside (IPTG)was added to the culture to induce CelA expression. Six hours later, thebacterial cells were collected by centrifugation at 10,000 g, 4° C. for15 min. After being washed with deionized water twice, the cells pelletwere resuspended in a sodium phosphate buffer (pH 7.4) and disrupted bysonication. Upon centrifugation at 15,000 g, 4° C. for 20 min, thesupernatant was collected and loaded onto a His-Trap affinitychromatography column (GE Health-care Bio-Sciences AB, Uppsala, Sweden).Fractions containing recombinant CelA (fused with a His-tag) were elutedwith a sodium phosphate buffer (pH 7.4) containing 200 mM imidazole and300 mM NaCl. Presence of CelA in these fractions was detected bySDS-PAGE analysis.

Characterization of CelA

(1) Enco-Glucanase Activity

The endo-glucanase activity of CelA was determined by CMC zymogramanalysis as described below. CelA was denatured by heating for 5 min at100° C. in a solution containing 1% (w/v) SDS and 2% (w/v) DTT and thenresolved on a 10% SDS-PAGE gel containing 0.2% (w/v) CMC. Afterelectrophoresis, the gel was washed three times (each for 30 min) with10 mM pH 8.0 Tris-HCl buffer containing 1% Triton X-100 and then soakedin the same buffer overnight to allow protein renaturation. Afterwards,the gel was incubated at 65° C. in 50 mM sodium acetate buffer (pH 5.0)for 30 min, stained with 0.2% (w/v) Congo red for 20 min, and destainedwith 1 M NaCl. A clear band against a red background was observed at theposition corresponding to CelA, indicating that CelA possessesendo-glucanase activity.

The endo-glucanase activity of CelA was also determined following themethod described in Miller et al., Anal Chem 31:426-428, 1959, undervarious conditions (i.e., temperature and pH value) to determine theoptional reaction condition of this enzyme. Briefly, 0.5 ml of asolution containing CelA was mixed with 0.5 ml of 1.0% CMC in 50 mMsodium phosphate buffer at a test pH (i.e., pH 4-9). The mixture wasincubation at a test temperature (i.e., 45, 55, 60, 65, 70, or 75° C.)for 15 min and the concentration of the reducing sugars (converted fromCMC by CelA) was determined using the well-known dinitrosalicylic acid(DNS) method. One unit of enzyme activity refers to production of 1 mmolglucose per minute. The protein concentration of the CelA solution wasdetermined by the Bradford method (see Bradford, Anal Biochem72:248-254, 1976) using a Bio-Rad Protein Assay Kit. As shown in FIG.2A, CelA was active in a broad temperature, i.e., 45-75° C.).

To determine its thermo-stability, CelA was incubated at 45, 55, 60, 65,70 or 75° C. for 6 hours and its endo-glucanase activity was analyzedafterwards using 1.0% CMC in 50 mM sodium acetate buffer (pH 5.0). Over80% of its endo-glucanase activity was maintained after 4-hourincubation at 75° C. (see FIG. 2B), indicating that CelA is athermophilic enzyme.

The endo-glucanase activity of CelA was determined at various pH values,i.e., pH 4-5 (in sodium acetate buffer) and pH 6-9 (in sodium phosphatebuffer). Further, CelA was incubated under various pH conditions (4-9)for 16 hours and its enzymatic activity was determined afterwards toexamine its stability under different pH conditions. As shown in FIG. 3,CelA exhibits endo-glucanase activity in a broad pH range, i.e., pH 4-9(see panel A) and remains stable after 16-hour incubation under thedifferent pH conditions (see panel B).

The endo-glucanase activity of CelA was determined in the presence ofvarious chemicals, i.e., CaCl₂, CoCl₂, CuCl₂, CuSO₄, EDTA, MgCl₂, MnSO₄,NaN₃, ZnSO₄, DTT, and 2-mercaptoethanol, to examine the impact of thesechemicals on CelA activity. Results indicate that certain divalentcations, i.e., Mn²⁺, Co²⁺, and Ca²⁺, stimulate CelA activity. See FIG.4.

(2) Substrate Specificity

The substrate specificity of CelA was analyzed at 65° C. using 1.0% ofthe various substrates listed below: Avicel, swollen Avicel, CMC,cellulose fiber, β-glucan (barely), filter paper, Lichenan,Xylan-brichwood and Xylan oat spelts. The results revealed that thisenzyme efficiently hydrolyzes amorphous substrates, including Avicel,CMC, β-glucan and Lichenan. (Table 1).

TABLE 1 Substrate specificity of Geobacillus sp. 70PC53 Cel A inincubation at 65° C. Specific activity Substrate (μmole glucose/mg/min)Avicel 0 Acid swollen Avicel 41.4 CMC 116.4 Cellulose fiber 0 β-glucan(barely) 1267.3 Filter paper 1.0 Lichenan 945.4 Xylan-Brichwood 5.3Xylan-oat spelts 0.1(3) Cellulolytic Activity Comparison Between CelA and Trichoderma reeseiCelluloses

Cellulases from Trichoderma reesei (ATCC 26921), substrates4-nitrophenyl b-D-glucopyranoside (pNPG), 4-nitrophenyl b-D-cellobioside(pNPC) and 4-nitrophenyl b-D-cellotrioside (pNPT) were purchased fromSigma. The enzymatic activity of T. reesei cellulases and CelA wereanalyzed at a concentration of 1.0 mM using the different substrateslisted above under at 37° C. and 65° C., respectively. 1 U of enzymeactivity was defined as production of 1 μmol 4-nitrophenol under thereaction conditions described above.

As compared with T. reesei celluloses, CelA exhibited a tenfold greaterenzymatic activity when CMC, pNPT and pNPC (all soluble) were used assubstrates. See Table 2 below.

TABLE 2 Comparison of enzyme activity between CelA and Trichodermareesei celluloses Cel A T. reesei Celluloses Substrate (U/mg, 65° C.)(U/mg, 37° C.) CMC 116.44 19.11 Avicel 0.71 3.84 Filter paper 1.06 19.13pNPT 84.48 7.87 pNPC 166.89 13.61 pNPG 0.35 11.56 β-1,3 Glucan (from1.08 0.23 Euglena gracilis)

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence at least 80% identical to SEQ ID NO: 1, wherein the isolatedpolypeptide has endo-glucanase activity.
 2. The isolated polypeptide ofclaim 1, wherein the amino acid sequence is at least 90% identical toSEQ ID NO:
 1. 3. The isolated polypeptide of claim 2, wherein the aminoacid sequence is at least 95% identical to SEQ ID NO:
 1. 4. The isolatedpolypeptide of claim 3, wherein the amino acid sequence is at least 99%identical to SEQ ID NO:
 1. 5. The isolated polypeptide of claim 4,wherein the amino acid sequence is SEQ ID NO:
 1. 6. The isolatedpolypeptide of claim 1, wherein the polypeptide has the amino acidsequence of SEQ ID NO:
 1. 7. A method of producing a fermentable sugarfrom a lignocellulosic material, comprising providing a multi-enzymecomposition containing an endo-glucanase that includes an amino acidsequence at least 80% identical to SEQ ID NO: 1, an exo-glucanase, and aβ-glucosidase, and contacting the multi-enzyme composition with alignocellulosic material to produce a fermentable sugar.
 8. The methodof claim 7, wherein the fermentable sugar is selected from the groupconsisting of glucose, xylose, arabinose, galactose, mannose, rhamnose,sucrose, and fructose.
 9. The method of claim 7, wherein theendo-glucanase has the amino acid sequence of SEQ ID NO:1.
 10. Themethod of claim 7, wherein the lignocellulosic material is selected fromthe group consisting of cellulosic animal waste, municipal solid waste,waste paper, yard waste, an agricultural residue, and a forestryresidue.
 11. A method of producing a product from a lignocellulosicmaterial, comprising providing a multi-enzyme composition containing anendo-qlucanase that includes an amino acid sequence at least 80%identical to SEQ ID NO: 1, an exo-glucanase, and a □-glucosidase,contacting the multi-enzyme composition with a lignocellulosic materialto produce a sugar, and converting the sugar to a product.
 12. Themethod of claim 11, wherein the converting step is performed bymicroorganism fermentation.
 13. The method of claim 11, wherein theconverting step is performed by enzyme treatment.